Cell surface oligosaccharides are known to play a crucial role in mediating cell-cell interactions in development and in the disease state. The developmentally-regulated patterns of glycoprotein glycosylation are determined largely by the activity and specificity of glycosyltransferase enzymes expressed in the Golgi (Rademacher, T. W. et al, Ann. Rev. Biochem. 57:785, 1988; and Yousefi, S. et al, J. Biol. Chem. 266:1772, 1991). Changes in glycosyltransferase activities have been associated with malignancies and other disease conditions, although the factors and intracellular signalling pathways which regulate expression of glycosyltransferase activities in the Golgi are largely unknown.
Several disease states are known to be associated with expression of specific glucosaminyltransferases resulting in altered patterns of cellular oligosaccharides. For example, the modification of cell surface carbohydrates has been linked to transformation and metastasis (Dennis, J. W. et al, Science, 1987). Malignant transformation of murine and human cells is commonly associated with expression of the larger complex N-linked oligosaccharides and increased polylactosamine content (Warren, L. et al. Biochem. Biophys. Acta. 516:97, 1978). Recent evidence suggests that the branching, extension and polylactosamine content of O-linked oligosaccharides may also affect metastasis or tumor growth (Yousefi S. et al, J. Biol. Chem. 266:1772, 1991).
The Golgi enzyme UDP-GlcNAc:Gal.beta.1-3GalNAc-R .beta.1-6-N-acetylglucosaminyltransferase, D-N-acetylglucosamine to D-N-acetylgalactosamine, (GlcNAc to GalNAc) (core 2 GlcNAc-T) substitutes "core 1" O-linked glycans (i.e., Gal.beta.1-3GalNAc.alpha.) to produce "core 2" structures (i.e., fold Gal.beta.1-3GalNAc.beta.1-6GlcNAc.alpha.). UDP-Gal: GlcNAC-R .beta.1-4-galactosyltransferase. .beta.1-4Gal-T subsequently acts on core 2 producing .beta.1-6 linked lactosamine which can be extended into polylactosamine by UDP-GlcNAc:Gal.beta.4GlcNAc-R.beta.3-N-acetylglucosaminyl (.beta.1-3GlcNAc-T(i) and .beta.1-4Gal-T. Core 2 GlcNAc-T activity appears to be an important rate limiting step in the extension of O-linked oligosaccharides with polylactosamine (i.e., repeating Gal.beta.1-4GlcNAc.beta.1-3), a structure which has been associated with malignant transformation (Yousefi et al, J. Biol. Chem. 266:1772, 1991).
Changes in the activity of core 2 GlcNAc-T have also been associated with the Wiskott-Aldrich immunodeficiency syndrome (WAS). Increased core 2 GlcNAc-T activity is closely associated with activation of human T cells in vitro, via the T cell receptor complex (Piller, F. et al, J. Biol. Chem. 263:15146, 1988). Furthermore, lymphocytes of patients with WAS show abnormal regulation of the enzyme (Higgins, L. A. J. Biol. Chem. 266:6280, 1991).
Core 2 GlcNAc-T activity appears to be regulated by factors which have an impact on intracellular signalling and developmental status of the cell. For example, T cell activation via the T cell receptor complex in vitro is associated with a 3 fold increase in core 2 GlcNAc-T activity (Piller, F. et al, J. Biol. Chem. 263:15146, 1988; and Higgins, L. A. et al, J. Biol. Chem. 266:6280, 1991). Lymphocytes from patients with WAS show both abnormal proliferative responses and abnormal regulation of core 2 GlcNAc-T. Enzyme activity is increased following transformation of rat 2 fibroblasts and murine mammary carcinoma cells by activated H-ras (Yousefi, S. et al., J. Biol. Chem. 266:1772, 1991), in human leukemias (Brockhausen, I. et al Cancer Res. 51:1257, 1991).
Tissue-specific patterns of oligosaccharide processing may be regulated at both the level of transcription of glycosyltransferase genes and post-translational modifications of their protein products. For example, recent studies of .alpha.2-6SA-T showed that mRNA levels varied between tissues by as much as 50 fold, correlating with tissue specific differences in enzyme activity (Paulson, J. C. et al J. Biol. Chem. 264, 1989). Tissue-specific regulation of glycosyltransferases may also take the form of alternate mRNA splicing, or alternate translation initiation and termination signals allowing production of multiple proteins, possibly with different activities or acceptor specificities. In this regard, 5 exons of the rat .alpha.2-6SA-T gene are present in liver transcripts while only 3 of these exons are present in kidney mRNA. The hepatic SA-T mRNA is transcribed from the first exon, while the kidney transcript is initiated from a promoter and ATG codon within the third intron (Wang, X et al Glycobiology 1:25, 1990). Tissue-specific promoter elements have also been identified in the rat .alpha.2-6SA-T gene which appear to control expression of the enzyme in both tissues (Paulson, J. C. et al., J. Biol. Chem. 264, 1989).
Studies of the role of glucosaminyltransferases in the oncodevelopmental and disease process have been limited by the lack of highly sensitive and specific assays for the individual transferase enzymes. Assays for glycosyltransferases have involved reacting a sugar nucleotide donor which is labelled with .sup.3 H or .sup.14 C and subsequently measuring the radioactivity of the acceptor to which the labelled sugar is transferred by the glycosyltransferase. Morito, in European Patent Application number 87118665.6, discloses a method for measuring glycosyltransferase using a constitution containing a donor which is not labelled and a substance which is specifically bound only to a product.